Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

Abstract

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic α-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally “unwound.” The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics. Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic α-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally “unwound.” The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics. The parathyroid hormone receptor (PTH1R) 3The abbreviations used are: PTH1RPTH/PTHrP receptorPTHparathyroid hormonePTHrPparathyroid hormone-related proteinGTPγSguanosine 5′-3-O-(thio)triphosphateECDextracellular domainBSAbovine serum albuminMBPmaltose-binding proteinPEGpolyethyleneglycolTMtransmembraneMOPS4-morpholinepropanesulfonic acidR.m.s.root mean-squaredR0PTH1R conformation in cell membranes in the presence of GTPγSRGPTH1R conformation in cell membranes in the presence of a high affinity, dominant negative Gαs protein. is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) (Ref. 1Jüppner H. Abou-Samra A.B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1156) Google Scholar; reviewed in Ref. 2Gardella T.J. Juppner H. Rev. Endocr. Metab. Disorders. 2000; 1: 317-329Crossref PubMed Scopus (47) Google Scholar). PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels (reviewed in Refs. 3Murray T.M. Rao L.G. Divieti P. Bringhurst F.R. Endocr. Rev. 2005; 26: 78-113Crossref PubMed Scopus (249) Google Scholar, 4Potts J.T. J. Endocrinol. 2005; 187: 311-325Crossref PubMed Scopus (258) Google Scholar, 5Potts J.T. Gardella T.J. Ann. NY Acad. Sci. 2007; 1117: 196-208Crossref PubMed Scopus (99) Google Scholar), to act on bone and kidney cells and thus restore blood calcium to normal levels. In bone, PTH directly stimulates osteoblasts, resulting in bone formation (reviewed in Ref. 6Jilka R.L. Bone. 2007; 40: 1434-1446Crossref PubMed Scopus (566) Google Scholar), which in turn activate osteoclasts to induce bone resorption. In the kidney, PTH stimulates the reabsorption of filtered calcium, inhibits the reabsorption of phosphate, and stimulates the synthesis of 1,25-dihydroxyvitamin D3. The paradoxical anabolic/catabolic actions of PTH on bone can be modulated by exogenous PTH, and provide the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (7Neer R.M. Arnaud C.D. Zanchetta J.R. Prince R. Gaich G.A. Reginster J.Y. Hodsman A.B. Eriksen E.F. Ish-Shalom S. Genant H.K. Wang O. Mitlak B.H. N. Engl. J. Med. 2001; 344: 1434-1441Crossref PubMed Scopus (4009) Google Scholar). Anabolic PTH therapy requires intermittent administration to minimize bone-resorptive effects, which predominate with sustained administration of PTH. PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hypercalcemia of malignancy (8Burtis W.J. Wu T. Bunch C. Wysolmerski J.J. Insogna K.L. Weir E.C. Broadus A.E. Stewart A.F. J. Biol. Chem. 1987; 262: 7151-7156Abstract Full Text PDF PubMed Google Scholar, 9Moseley J.M. Kubota M. Diefenbach-Jagger H. Wettenhall R.E. Kemp B.E. Suva L.J. Rodda C.P. Ebeling P.R. Hudson P.J. Zajac J.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5048-5052Crossref PubMed Scopus (702) Google Scholar, 10Suva L.J. Winslow G.A. Wettenhall R.E. Hammonds R.G. Moseley J.M. Diefenbach-Jagger H. Rodda C.P. Kemp B.E. Rodriguez H. Chen E.Y. et al.Science. 1987; 237: 893-896Crossref PubMed Scopus (1189) Google Scholar, 11Klein R.F. Strewler G.J. Leung S.C. Nissenson R.A. Endocrinology. 1987; 120: 504-511Crossref PubMed Scopus (22) Google Scholar) and was subsequently shown to be a critical developmental paracrine factor that controls endochondral bone formation (Refs. 12Karaplis A.C. Luz A. Glowacki J. Bronson R.T. Tybulewicz V.L. Kronenberg H.M. Mulligan R.C. Genes Dev. 1994; 8: 277-289Crossref PubMed Scopus (1015) Google Scholar, 13Lanske B. Karaplis A.C. Lee K. Luz A. Vortkamp A. Pirro A. Karperien M. Defize L.H. Ho C. Mulligan R.C. Abou-Samra A.B. Jüppner H. Segre G.V. Kronenberg H.M. Science. 1996; 273: 663-666Crossref PubMed Scopus (1148) Google Scholar; reviewed in Ref. 14Kronenberg H.M. Ann. NY Acad. Sci. 2006; 1068: 1-13Crossref PubMed Scopus (350) Google Scholar). PTHrP can also mediate bone-anabolic effects when administered to osteoporosis patients (15Horwitz M.J. Tedesco M.B. Gundberg C. Garcia-Ocana A. Stewart A.F. J. Clin. Endocrinol. Metab. 2003; 88: 569-575Crossref PubMed Scopus (173) Google Scholar) and has been suggested to be more anabolic than PTH due to a differential effect on the coupled bone formation and resorptive responses (16Plotkin H. Gundberg C. Mitnick M. Stewart A.F. J. Clin. Endocrinol. Metab. 1998; 83: 2786-2791Crossref PubMed Scopus (57) Google Scholar). PTH/PTHrP receptor parathyroid hormone parathyroid hormone-related protein guanosine 5′-3-O-(thio)triphosphate extracellular domain bovine serum albumin maltose-binding protein polyethyleneglycol transmembrane 4-morpholinepropanesulfonic acid root mean-squared PTH1R conformation in cell membranes in the presence of GTPγS PTH1R conformation in cell membranes in the presence of a high affinity, dominant negative Gαs protein. PTH and PTHrP are encoded by separate genes, each of which is found in vertebrate species ranging from fish to man. How PTH and PTHrP evolved to mediate distinct biological activities: calcium/phosphate homeostasis and tissue development, respectively, via actions upon a single receptor, remains unclear. Amino acid sequence homology is most apparent in the first 34-residue segments of the proteins, and N-terminal 34-residue peptide fragments of PTH and PTHrP are sufficient for high affinity binding to the PTH1R and are generally found to be equally potent for stimulating cAMP formation in PTH1R-expressing cells (1Jüppner H. Abou-Samra A.B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1156) Google Scholar). The interaction of the (1–34)-length ligand with the PTH1R has been postulated to follow a “two-domain” model: residues within the approximate (1–14) segment interact with the 7-transmembrane (7-TM) helical domain embedded in the membrane, and residues within the approximate (15–34) segment interact with the N-terminal extracellular domain (ECD) of the receptor (17Bergwitz C. Gardella T.J. Flannery M.R. Potts Jr., J.T. Kronenberg H.M. Goldring S.R. Jüppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 18Luck M.D. Carter P.H. Gardella T.J. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar). The 1–14 domains of PTH and PTHrP share eight amino acid sequence identities, reflecting a critical role in activating the receptor (18Luck M.D. Carter P.H. Gardella T.J. Mol. Endocrinol. 1999; 13: 670-680Crossref PubMed Google Scholar), while the 15–34 domains share amino acid identities, a critical role in high affinity binding to the receptor. studies that PTH and PTHrP in their capacities to to two PTH1R conformations T. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. PubMed Scopus Google Scholar, T. A. M.J. M. Jüppner H. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. 2006; PubMed Scopus Google Scholar, S.R. Gardella T.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. Potts Jr., J.T. Gardella T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is in the presence of but in the of G protein with signaling responses in and in and is bound by The is to GTPγS by the of a high affinity of and bound by basis for the capacities of PTH and PTHrP ligands to to altered PTH1R conformations is the (1–14) and (15–34) of PTH to the to to the conformation T. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. PubMed Scopus Google Scholar, S.R. Gardella T.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. Potts Jr., J.T. Gardella T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a that to the high resolution crystal structure of PTH1R ECD in with the 15–34 of PTH Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The PTH1R ECD a that is class B C. M. P. R. S. J. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar, K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. K. J. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the domain binds as a continuous and amphipathic α-helix to a hydrophobic groove in the the high resolution crystal structure of the PTHrP in with the PTH1R which reveals a distinct conformation the of the PTHrP peptide. on the hybrid PTH/PTHrP peptides for residues involved in altered ECD functional of peptides confirmed that the altered modes of binding into functional consequences in of receptor These results provide critical into how PTH and PTHrP can act through a single receptor, and a model for designing better PTH/PTHrP for PTH1R residues with a and a was from Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The was with and and into a maltose-binding protein by a and a sequence in in and the in The resulting the protein and The was by of the The protein used for which residues of PTH1R to the of was Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The protein was in B cells The cells were in to the was to was to and protein was with for a of The cells were by and the protein was as for the protein Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the suggested that the protein was as by two distinct of different The protein was by the of PubMed Scopus Google Scholar). for ECD binding studies and were and by hybrid peptides were and by the The interaction PTH1R ECD and and was by the was from the ECD protein by with a of to protein in with was by binding were with the to by a and in with to of the and establish a The peptides were in with and their and were for each for with peptide and PTH1R ECD were from the with the peptides and PTH1R In a was a from a that PTH1R but peptide. was used to the The binding of peptides to the protein was with an The were and each of and and each of and in a of and The PTH and were for to the and The were peptides were as and the were to were in an was used to the to a for of in and was with a PTHrP a on for and to for were by the with a of The were into a of by and were by into was from a single crystal of the a of and of K. The PubMed Scopus Google Scholar) was used to the and the were to structure factor with the 1994; PubMed Scopus Google Scholar). was of in the The structure was by molecular with M.D. J. 2007; 40: PubMed Scopus Google Scholar) separate for and the PTH1R ECD from the Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is in the of in G.J. PubMed Scopus Google Scholar) and with PubMed Scopus Google Scholar) the M.D. 2001; PubMed Scopus Google Scholar) was two to the and the with R.A. J.M. J. 26: Google Scholar) that of the residues were in the most of the and residues were in the The and are in of and were performed within the were with and crystal was in are for the low in the is due to in the The are in the crystal was in are for the The low in the is due to in the The are in the in a binding to the G receptor and G protein-coupled receptor conformations in cell membranes was as M. S. Potts Jr., J.T. Gardella T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of cAMP in cells PTH1R was performed in tissue as L.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). cAMP was with a cAMP to the first the binding of PTH and PTHrP ligands to the protein an In the is bound to and the protein is bound via to of the ligand with the protein the into and thus a that inhibits with an of which was to the for the binding of to the protein by Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and each the interaction with an of in with bound the ECD with an affinity than that of the C-terminally peptide binding affinity can be by the crystal structure of the PTH1R which that two are the ECD and the ligand Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In and the with of a is for high affinity binding of PTHrP to the also the interaction of the peptides with the isolated ECD the which to binding in The isolated PTH1R ECD of and a single the was on the of and the binding of and in was of the binding that PTH and PTHrP bound the ECD with of and and The for PTH binding to the ECD is to that by U. H. K. M. A. R. 2000; PubMed Scopus Google Scholar). The binding that PTH and PTHrP the PTH1R ECD with but in the the of PTH, but to the receptor binding of the crystal structure of the protein in with The structure was by molecular and resolution to an factor of factor was for PTH1R residues segments and the which were from the The PTH1R ECD the class B ECD Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and the PTHrP ligand an amphipathic α-helix that the hydrophobic groove in the ECD the of the N-terminal the two and the α-helix and was for PTHrP residues The is by hydrophobic interactions PTHrP residues and which the hydrophobic groove of the ECD by residues Ile-115, and B and residues are with a to from receptor In an of the peptide and and The an with an to the of and with the side chain of The PTHrP helix the the formation of the of and the of the side chain of and the of and of and the of and the of and with binding in the of PTHrP interactions with the a with but interaction be by a crystal interaction that the of and that in the contacts the domain of the receptor R.C. N. J. Gardella T.J. Mol. Endocrinol. 2003; PubMed Scopus Google Scholar). of the with the and in the binding of the two peptides In the hydrophobic of the amphipathic peptide binds the hydrophobic groove in the and the interaction is by the residues and The most the of the PTH a continuous α-helix from to the to the In the PTHrP helix from to which and the is involved in receptor The PTHrP helix gently that helical from that of PTH due to the in residues and of the peptides and The of the of with for the The peptide and as by the J. Mol. Biol. PubMed Scopus Google Scholar) is for the than for the the PTH peptide more and the ECD with than the peptides for the of the by residues of PTHrP The receptor accommodates the different binding modes of the two peptides with in conformation. The of the two are a of their of in the two is for the side chain conformations of Leu-41 and In is apparent Leu-41 and of the ECD and and of the In the structure the rotamer S. J. Mol. Biol. 2001; PubMed Scopus (22) Google Scholar) the of Leu-41 and the of In in the structure Leu-41 the the receptor to accommodate the side chain of and the of Leu-41 and the of Leu-41 rotamer toggle switch was in an by and R. M.J. Mol. PubMed Scopus Google Scholar) on and the same rotamer conformation in the and but the side chain the of the ECD in the thus the of the PTHrP helix the of the peptide binding of the ECD to the different binding modes of PTH and PTHrP by the conformation of residues Leu-41 and the different binding modes in the crystal the of PTH and PTHrP residues to ECD binding by the of of and to the interaction of PTH with ECD in the shown Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), PTH residues and were most critical for ECD with by and PTHrP residues and were for ECD with and the most critical contacts and of PTHrP are as critical for ECD binding as and of PTH. and of PTHrP are more for ECD binding than and of PTH. These in binding results are with the binding modes in the crystal to which residues were responsible for the ECD binding modes of PTH and The crystal that the residues and of the peptides their ECD binding that the residues in peptide to the residues in the peptide the PTH and PTHrP binding was by the binding of hybrid peptides of the PTH/PTHrP residues to the ECD protein in the The hybrid peptides were in the and and a single of the a two of the a In the adopt the binding of the in of ECD binding affinity, and to to the binding mode used by the The peptide was of the PTHrP residues and to a as by the ECD-binding of the and peptides In the normal binding affinity, and single was to the by the and as shown by the binding of the and The of in the peptide in as with that the in the PTH and PTHrP binding modes are by more than within the of the PTH The effect of the in PTH PTH the PTHrP binding but the side of and of PTH, by and in respectively, the ECD-binding affinity of and that the PTH peptide to adopt the PTHrP ECD binding normal ECD-binding when altered with the PTH and but binding with the The of the was by the and by the in the and The PTHrP peptide normal binding to the with the that peptide the PTH binding the C-terminally ECD affinity In the ECD affinity when C-terminally but ECD affinity when C-terminally that are to PTHrP to adopt the PTH ECD-binding to the ECD binding affinity of PTH The most of the in and B is the functional of and that is for In the of and in ECD while the and were The is that the as hydrophobic as the and The in PTH in hydrophobic to the receptor of the side and be by the contacts from a the in PTHrP in hydrophobic to the receptor the The be by the hydrophobic contacts from a the effects of the ligand on the of the ligands to to the PTH1R in cell first binding in performed as a for which binding on interactions to the receptor and in the presence of which the G PTH1R T. Potts Jr., J.T. Gardella T.J. Mol. Endocrinol. PubMed Scopus Google Scholar, M. S. Potts Jr., J.T. Gardella T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that in binding to the isolated ECD also in binding to the PTH1R binding in performed as a which binds to the receptor transmembrane domain and membranes from cells that the receptor with a high affinity of as to the G protein-coupled receptor conformation The same that binding to effect on binding to and and and were in as peptides in binding to the ECD as as to the but normal affinity for the conformation. In the effects of on the isolated were also apparent on the PTH1R and These results that the binding interactions by the ECD a role in the of the ligand to to the PTH1R in the but to the PTH1R in the conformation. with a more role for the in transmembrane peptides that affinity for but high affinity for high for stimulating cAMP in cells PTH1R of cAMP signaling by hybrid cells PTH1R were with the peptides for which the cells were and cAMP was The are the of and are to the cAMP in the presence of for which cAMP was and cAMP was The for PTH, and were and PTH and PTHrP distinct biological actions by activating their receptor, high resolution understanding of how the peptides the receptor is to the responsible for the of homeostasis and bone by PTH and bone by and also has for the design of peptide for In a structure of PTHrP in with the PTH1R with structure of the ECD Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), have more of the used by the PTH1R ECD to PTH and The peptides to the ECD as amphipathic that the hydrophobic groove in the with of the isolated peptides that structure in the 15–34 fragments of the ligands K. P. P. 2000; PubMed Scopus Google Scholar, M. E. A. P. 1999; PubMed Scopus Google Scholar). of the residues in the 15–34 and the interaction with the the peptides that their different contacts to the ECD a by ECD-binding that the of PTH, but is for ECD binding The in the 15–34 a critical to the receptor in the but is in the and are in a peptide that the PTH receptor S.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar), but is with that the N-terminal of the 15–34 fragments the of the interactions and the in their interaction with the receptor. The receptor accommodates the two different peptides with in the ECD a in the side chain in response to the PTHrP and a rotamer toggle switch in the Leu-41 side chain to with the side chain ligand and PTHrP was generally more to than PTH with to ECD of the single of PTHrP normal binding to the but of the PTH single the The PTH interaction is more to the of to the ECD and which for to the in response to that PTHrP provide a better in which to for The of the ECD affinity of PTH/PTHrP peptides with their selectivity has for the of PTH/PTHrP for studies on PTH that the of a ligand to to the receptor conformation with the of calcium and responses in as as with the effects on bone M. S. Potts Jr., J.T. Gardella T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). which is more to be a more anabolic than PTH with side effects of hypercalcemia (16Plotkin H. Gundberg C. Mitnick M. Stewart A.F. J. Clin. Endocrinol. Metab. 1998; 83: 2786-2791Crossref PubMed Scopus (57) Google Scholar). The of the high resolution design of the that peptide that are selective for the G protein-coupled receptor, and thus have more in of as the signaling and bone-resorptive effects with PTH M. S. Potts Jr., J.T. Gardella T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in studies and in studies be to S. and J. for with of the for on the and for on the cAMP with